The invention relates to the fields of cell enrichment, blood sampling, and microfluidic devices.
Lab-on-a-chip technologies for cell-based, basic scientific or clinical applications promise to integrate all procedures from primary sample collection to data analysis in small, inexpensive, and versatile devices (Andersson and Van Den Berg, Sensors and Actuators B-Chemical, 2003, 92, 315-325; Sia and Whitesides, Electrophoresis, 2003, 24, 3563-3576; Toner and Irimia, Annual Review of Biomedical Engineering, 2005, 7, 77-103). One step in this complex process is sample preparation, when cells from primary samples are typically separated, washed, and re-suspended in new buffer solutions, with or without specific stimulation steps, before they are made available for subsequent processing and analysis. However, most of the time this step is accomplished not on the chip but on the bench, by procedures like pipetting and centrifugation, because no common, easy-to-implement approaches exist today for on chip sample preparation.
Handling of mammalian cells in microfluidic devices poses some nontrivial challenges. Eukaryotic cells in general and human cells in particular are mechanically more fragile and more deformable than other cells. They are also biologically more sensitive and quicker to respond to changes in their environment. While various methods for handling cells in suspensions have been proposed, each technique has drawbacks that limit its potential. Methods using electric fields for trapping and exposing mammalian cells to new reagents (Rosenthal and Voldman, Biophysical Journal, 2005, 88, 2193-2205; Gascoyne and Vykoukal, Proceedings of the Ieee, 2004, 92, 22-42; Seger et al., Lab on a Chip, 2004, 4, 148-151) are dependent on the solution for cell suspension and on the cell type. Optical manipulation of relatively large mammalian cells (Arai et al., Electrophoresis, 2001, 22, 283-288) can be laborious and expensive and cannot be easily scaled up, while the use of mechanical structures (Panaro et al., Biomolecular Engineering, 2005, 21, 157-162; Wheeler et al., Analytical Chemistry, 2003, 75, 3581-3586; Glasgow et al. Wheeler, Ieee Transactions on Biomedical Engineering, 2001, 48, 570-578) is usually irreversible, since once cells are mechanically trapped they cannot be easily released.
Precise metering of whole blood samples is essential for many clinical diagnostic applications, e.g., the biochemical analysis of blood (Tudos et al., Lab on a Chip, 2001, 1, 83-95) and blood cell counting and analysis (Toner and Irimia, Annual Review of Biomedical Engineering, 2005, 7, 77-103). Volumes of blood as small as a few microliters can be precisely sampled using syringes and micropipettes. However, smaller volumes, like those used in microfabricated devices require different approaches not always suited to complex cell-rich fluids like blood. Vented capillaries with a hydrophobic barrier (Pugia et al., Clinical Chemistry, 2005, 51, 1923-1932; Ahn et al., Proceedings of the Ieee, 2004, 92, 154-173; Columbus and Palmer, Clinical Chemistry, 1991, 37, 1548-1556) could trap air bubbles between fluid segments that need to be mixed. Microdroplets on electrowetting platforms (Srinivasan et al., Lab on a Chip, 2004, 4, 310-315) have to be formed outside the device by pipetting; their minimum size is limited to the microliter range, and stickiness of blood proteins and cells on the hydrophobic surfaces may be problematic. Finally, valves have been designed to sample sub-microliter volumes of fluid and perform biochemical assays (Hansen et al., Proceedings of the National Academy of Sciences of the United States of America, 2002, 99, 16531-16536), although the geometry of the valves is not friendly for cells. Precise metering volumes are possible using valves in channels with vertical walls (Li et al., Electrophoresis, 2005, 26, 3758-3764).
Thus, there is a need for new devices and methods for manipulating samples in microfluidic devices.